Papillomaviruses are small DNA viruses that infect a variety of animal species. Some are associated with the development of malignancies in their natural hosts. Over 60 types of human papillomavirus (HPV) have been identified. These infect humans at a variety of body locations and are responsible for common skin warts, laryngeal papillomas, genital warts and other wart-like lesions. Genital HPV infections are particularly common and a number of HPV types, but most frequently types 6, 11, 16 and 18, infect the genital tract in both men and women. In women, HPVs infect various portions of the genital tract including the cervix.
Genital HPVs are a significant clinical problem. HPV infection of the ano-genital region is now regarded as the most common form of viral sexually-transmitted disease (STD). The viruses cause genital infections which become manifest in one of three ways:
i clinical infection, where gross genital warts are visible; PA1 ii subclinical infection, where viral lesions are not obvious but are detectable using special viewing techniques; and PA1 iii latency, where the only sign of infection is the presence of HPV DNA. PA1 (i) use whole proteins (this introduces the possibility that contaminating DNA may be associated with the proteins); PA1 (ii) use point mutants (this can lead to reversion to native protein, which requires multiple mutations to avoid; in addition, any point mutation leads to loss of potentially vital epitopes); PA1 (iii) use specific peptides (this requires a very large number of peptides, the identification of which is very complex, to make a vaccine of broad utility); and PA1 (iv) use variants such as fusions and combinations of deletion mutants (this method has none of the above limitations).
Subclinical infections are common. It is estimated that 2 to 4% of Papanicolaou (Pap.) smears show evidence of HPV. Latent infections are even more frequent and the majority of adults harbour one or more types of genital HPV.
Carcinoma of the uterine cervix (CaCx) is a common cancer in women. Two forms of cervical cancer are recognised; squamous cell carcinoma (SCC) is by far the most frequent representing about 90% of observed cases; adenocarcinoma, a cancer of the secretion cells, accounts for about 10%. Cancer of the cervix develops through pre-cancerous intermediate stages to invasive forms (the carcinoma) which can become life threatening. The pre-cancerous stages of increasing severity are known as cervical intraepithelial neoplasia (CIN) grades 1 to 3. Over a 20 year period about 40% of the untreated CIN3 patients develop invasive cancer, the increasingly serious forms of which are known as stage I to IV. Invasive cancer frequently leads to death.
Cervical cancer in both its pre-cancerous and invasive stages is one of the few cancers for which a highly reliable and relatively cheap screening method is available. The Papanicolaou (Pap.) smear involves cytological examination of cervical scrapes to test for the presence of abnormal cervical cells which are indicative of pre- or invasive cancer. Detection of abnormalities leads to further investigation and treatment if necessary.
To be effective at reducing the number of cervical cancers and resultant deaths, Pap. smear screening is undertaken on a mass scale and ideally includes all women of sexually-active age. Detection and subsequent treatment of CIN has a very high success rate in the prevention of invasive cancer, while early detection of the latter can have a marked effect on mortality.
Most developed countries have highly developed Pap. smear screening programs which have resulted in a 30% drop in age-specific mortality due to CaCx between 1960 and 1980. However, apart from the Scandinavian countries, few developed countries screen more than 50 to 60% of women, allowing CaCx and resultant deaths to remain a significant problem. In the developing world the situation is even worse as few organised screening programs exist, resulting in 400,000 new cases of invasive cancer annually in these countries.
As outlined earlier, a variety of types of HPV cause genital infections in humans, although four types (6, 11, 16, 18) predominate. Evidence collected over the past 15 years strongly suggests that several of the HPVs are associated with the development of cervical cancer. Indeed many researchers have concluded that specific HPV types are the essential aetiologic factor responsible for the development of many of the cancers.
Infection with HPV-16 and HPV-18 has been associated with the development of cancer of the cervix. It has been postulated that HPV acts as an initiator in cervical carcinogenesis and that malignant transformation depends on interaction with other factors. Infections with HPV-6 and HPV-11 have been associated with the development of genital warts. The incidence of HPV infection appears to be increasing as shown by a large increase recently in patient visits related to genital HPV infections in both males and females and the presence of HPV in Pap. smears of some women under 30 years of age.
The nature of HPV-16 in particular and papilloma viruses in general has been well studied recently. HPV-16 contains a 7904 bp double-stranded DNA genome (Siedorf, K. et al., Virology (1985) 145:181-185). The capsid is 50 nm and contains 72 capsomers (Klug, A., J. Mol. Biol. (1965) 11:403-423). U.S. Pat. No. 4,777,239 discloses a series of 17 synthetic peptides which are said to be capable of raising antibodies to HPV-16 and thus may be useful for diagnostic purposes. In addition, European Patent 0 412 762 discloses polypeptides which are antagonists of the biochemical interaction of the HPV E7 protein and the retinoblastoma gene (RBG) protein, and which are said to be useful in the treatment of genital warts and cervical cancer.
The DNAs of several papilloma viruses have been sequenced, including several HPV types, bovine papillomavirus (BPV) and cottontail rabbit papillomavirus (CRPV). All of these display similar patterns of nucleotide sequence with respect to open reading frames. The open reading frames can be functionally divided into early regions (E) and late regions (L); the E regions are postulated to encode proteins needed for replication and transformation; and the L regions to encode the viral capsid proteins (Danos, O., et al., J. Invest. Derm. (1984) 83:7s-11s).
Two HPV encoded proteins, E6 and E7, are thought to be involved in the pathogenesis of HPV-induced abnormal cell proliferation (reviewed in Stoppler et al., Intervirology, (1994) 37:168-179). The amino acid sequences of the HPV-16 E6 and E7 proteins as deduced from the nucleic acid sequence are shown in Siedorf et al., Virology, (1985) 145:181-185.
The HPV genes encoding the E6 and E7 proteins are invariably expressed in tissue or tumor cells obtained from cervical cancers associated with HPV infection. In addition, the HPV E6 and E7 genes derived from the HPV-16 strain are capable of inducing epithelial cell transformation in cell culture without the presence of other HPV genes. These observations indicate that at least part of the stimulation of cell proliferation caused by HPV infection is due to the E6 and E7 viral proteins.
The HPV E6 and E7 proteins are believed to be effective immunological targets for tumour regression. As described above, however, the E6 and E7 genes are known to "transform" cells possibly by the action of their protein products in interfering with cellular proteins involved in the control cell growth. Accordingly, if even minute traces of DNA encoding the E6 and E7 proteins were to be present in a vaccine preparation, this could cause that vaccine preparation to initiate irreversible transformation events in the cells of a recipient of the vaccine preparation. It is an object of the present invention to provide non-transforming variants of the HPV E6 and E7 proteins which are able to induce in a host animal (particularly a human) a range of humoral and cellular immune responses, and which are therefore suitable for use in the production of vaccines for the prevention, prophylaxis, therapy and treatment of HPV-induced diseases or other conditions which would benefit from inhibition of HPV infection.
In the work leading to the present invention, it has been recognised that there are four ways to induce immune responses to E6 and/or E7 proteins:
In addition to the cell transforming properties of the E7 protein itself, fusions of this protein with .beta.-galactosidase have also been shown to be cell-transforming (Fujikawa et al., Virology, 204, 789-793, 1994). Accordingly, it could not be predicted that fusions of E6 and/or E7 moieties, either full length or non-full length, would not also be cell-transforming.